The general potential of plant cystatins for the development of insect-resistant transgenic plants still remains to be established given the natural ability of several insects to compensate for the loss of active cysteine proteases following inhibitor ingestion. Here we assessed the potential of cystatins for the development of banana lines resistant to the banana weevil Cosmopolites sordidus, a major pest of banana and plantain in Africa. Protease inhibitory assays were first conducted with protein and methylcoumarin (MCA) peptide substrates to measure the inhibitory efficiency of different cystatins in vitro, followed by a diet bioassay with cystatin-infiltrated banana stem disks to monitor the impact of two plant cystatins, oryzacystatin I (OC-I, or OsCYS1)and papaya cystatin (CpCYS1), on the overall growth rate of young weevil larvae. As observed earlier for other Coleoptera, banana weevils produce a variety of proteases for dietary protein digestion, including in particular Z-Phe-Arg-MCA-hydrolyzing (cathepsin L-like) and Z-Arg-Arg-
MCA-hydrolyzing (cathepsin B-like) proteases active in mildly acidic conditions. Both enzyme populations were sensitive to the diagnostic cysteine protease inhibitor E-64 and to different plant cystatins including OsCYS1. In line with these broad inhibitory effects of cystatins, OsCYS1 andCpCYS1 caused an important growth delay in young larvae developing for 10 days in cystatininfiltrated banana stem disks. These promising results, which illustrate the natural susceptibility of
C. sordidus to plant cystatins, are discussed in the light of current genomic data on coleopteran cysteine cathepsins and recent hypotheses suggesting a key role for digestive cathepsin B-like enzymes as a determinant for resistance or susceptibility to plant cystatins in Coleoptera.Keywords: Banana weevil (Cosmopolites sordidus); banana (Musa spp.); cathepsin B-like proteases; cysteine cathepsins; plant cystatins; oryzacystatin I Plant cystatins against the banana weevil Page 3
INTRODUCTIONThe banana weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) is an insect pest of considerable importance in Africa, associated with the rapid decline of banana and plantain plantations (Gold, 1999a(Gold, , 2000Swennen and Vuylsteke, 2001;Gold et al., 2005). At the adult stage, banana weevil females deposit their eggs inside host plant tissues, at the base of the pseudostem or on an exposed corm. On hatching, the larvae tunnel through the corm to feed and develop, where they damage tissues, compromise water and mineral uptake, and weaken the colonized organs. Banana weevil infestations cause important losses in the field by a direct negative impact on harvestable bunch weight and a weakening effect on infested organs causing plant toppling during windstorms (Sengooba, 1986;Rukazambuga et al., 1998).Banana weevil control in Africa relies essentially on cultural and sanitation practices such as the use of clean planting material, the systematic trapping of adult weevils to prevent population build-up, an...
